PROJECT SUMMARY/ABSTRACT Chromosomal rearrangements are a fundamental form of mutagenesis with a profound impact on human health. Poorly understood processes of structural mutagenesis are active in somatic cells where they ultimately lead to cancer. A flood of genomic data from The Cancer Genome Atlas (TCGA) and other projects is revealing that intrachromosomal rearrangements are especially common and that certain genomic loci are highly prone to their occurrence. The nature and mechanisms of unstable loci are thus of central importance to cancer etiology. We are exploring these questions using models of acquired genomic copy number variants (CNVs), a term encompassing interstitial deletions and duplications and representing the same mechanisms as copy- number-neutral inversions and translocations. In these models, exogenous replication stress in the form of low- dose aphidicolin or hydroxyurea is a potent inducer of new CNVs in cultured somatic cells. CNVs are characterized by microhomologous junctions typical of pathogenic rearrangements that likely arise as replication errors. Hotspots of induced CNV formation are the same loci as common fragile sites, and it is the active transcription of large genes that leads to their extreme cell-type-specific instability. This project explores the hypothesis that the same instability mechanism(s) observed in these models of exogenous somatic CNV induction lead to recurrent genomic alterations in cancer as a result of endogenous replication stress. This idea is tested in three aims that examine the mechanisms leading to the extreme locus instability at large transcribed genes and the consequences of these mechanisms for the cancer genome. Aims 1 and 2 address non-exclusive hypotheses for how transcription interacts with replication stress to confer locus instability. Aim 1 argues that large genes create a dynamic conflict in which transcription into S-phase removes late-firing replication origins and creates large replicons highly sensitive to replication inhibition. Novel approaches will test this hypothesis by determining the cell-cycle timing of replication, transcription, and origin presence and firing. Aim 2 argues that transcription leads to persistent R-loops that cause fork stalling and thus precursor lesions for CNV formation. Monitoring and manipulating R-loop formation in cell lines that variably express specific large genes will test this hypothesis. Aim 3 addresses the consequences of these mechanisms on the cancer genome first through bioinformatic explorations of TCGA cancer data sets to correlate deletion hotspots with tumor- and cancer-type-specific transcription. Tissue-culture models of forced oncogene activation and mouse models of colon cancer will relate de novo CNV formation with the endogenous replication stress inherent to cancer.